This SBIR application seeks support to develop a Fluorescence Correlation Spectroscopy (FCS) platform for analysis of receptor-ligand interactions that is within the technical and financial reach of researchers and is adaptable to high throughput screening of chemical libraries. Precision Detectors Inc. has a long history in Photon Correlation Spectroscopy. Our QELS units determine the diffusion coefficients of macromolecules in solution by measuring fluctuations in scattered light resulting from their molecular motion. FCS is several orders of magnitude more sensitive than QELS and measures the molecular motion of single fluorescent molecules, macromolecules or multimolecular complexes as they diffuse into and out of approximately 1 fL volume defined by a confocal microscope. FCS detects both slow and rapid diffusional components present in a mixture and can thus quantitate free and receptor-bound ligands. At present, FCS is available only to those who can build their own instrument or who have several hundreds of thousands of dollars for instrument acquisition. Our aims are to make an instrument that is accessible to researchers and, because FCS is inherently miniaturized, to realize its potential for high throughput, nano-scale screening. Our initial approach is centered around a 682 nm laser diode. This laser is small, inexpensive, stable and is at least 10-fold more efficient than competing laser technologies. This excitation wavelength is also compatible with a new generation of photostable dyes (Alexa Fluor, Molecular Probes) that are bright and offer useful chemistries for conjugation with ligands and macromolecules. The platform is based on a flow cell/fixed objective system. Specific Aim 1 will optimize the lens system of the FCS to define and optimize confocal volume. Specific Aim 2 is to provide Peltier-temperature control. Specific Aim 3 is to develop a sample delivery route that minimizes sample volume and is adaptable to robotic solution delivery. Specific Aim 4 is to develop a software suite for instrument control, data collection and comprehensive analysis to obtain molecular concentrations, diffusion coefficients and free:bound ligand ratios. Overall system performance will be validated by using ligand/receptor systems (e.g. EGF/EGF-proteoliposomes; antiGluT1 IgGs/GluT1-proteoliposomes) in which ligands and receptors are well-characterized and show widely different diffusion coefficients. The availability of a platform permitting high throughput analysis of receptor/ligand binding thermodynamics at the single molecule level will be of great interest to the biomedical research community.